Molecular capsule comprising quercetin coated with polypeptides and preparation method therefor

Abstract

The present invention belongs to the technical field of molecules, and in particular relates to a molecular capsule comprising quercetin coated with polypeptides and a preparation method therefor. The method comprises: (1) mixing amino acid A, quercetin and water in proportion, and performing ultrasonic enzymolysis to prepare a precursor; (2) mixing the precursor and amino acid B in proportion to prepare a molecular capsule precursor; and (3) adding the molecular capsule precursor to a crystallizer for the recrystallization of an outer layer structure, and performing filtration and high-pressure drying to prepare the molecular capsule comprising quercetin coated with polypeptides. The present invention can effectively improve the bioavailability of quercetin in the constructed molecular capsule by optimizing the powder physical properties of drug crystals, and achieve controllable sustained release through a specific sphere diameter, thereby better controlling the release rate and release amount of quercetin.

Description

A polypeptide-coated quercetin molecular capsule and its preparation method Technical Field

[0001] The present invention belongs to the field of molecular technology, and in particular relates to a polypeptide-coated quercetin molecular capsule and a preparation method thereof. Background Art

[0002] Quercetin, also known as quercetin, quercetin, and quercetin, is a plant-derived flavonol and a flavonoid compound found in fruits, vegetables, and grains. Currently, there is no clear research demonstrating the therapeutic effects of quercetin. However, the article CLTJHAB, ALGPLP, CSAB, et al. Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease[J] demonstrates the potential value and utility of quercetin in clinical medicine, further garnering attention for its research and utilization.

[0003] In recent years, articles such as Stewart LK, Soileau JL, Ribnicky D, et al. Quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in C57BL / 6J mice fed a high-fat diet.[J]. have also shown that it has great prospects for use on the surface of the body and can achieve anti-inflammatory and metabolic effects.

[0004] However, current research has found that quercetin has significant drawbacks. The main drawback is that quercetin has a high release rate but a small release depth, resulting in a low actual effective utilization rate.

[0005] The current mainstream research direction is to construct container molecules to encapsulate quercetin, forming molecular capsules. Specifically, reversible supramolecular capsules are constructed through non-covalent interactions such as hydrogen bonding, electrostatic interactions, halogen bonding, hydrophobic interactions, and metal coordination bonds. However, existing approaches to preparing quercetin-encapsulated molecular capsules suffer from technical deficiencies such as low drug loading, poor bioavailability, high drug irritation, and slow effective drug release. Summary of the Invention

[0006] In order to solve the defects of existing quercetin use and the problems of low drug loading, poor bioavailability, strong irritation and poor release controllability of existing quercetin molecular capsules, the present invention provides a polypeptide-coated quercetin molecular capsule and a preparation method of the molecular capsule.

[0007] The main objectives of the present invention are:

[0008] 1. It can improve the biosafety of quercetin molecular capsules and reduce irritation;

[0009] Second, it can increase the drug loading capacity of quercetin molecular capsules;

[0010] 3. Achieve controlled release to improve the actual utilization effect of quercetin capsules.

[0011] To achieve the above objectives, the present invention adopts the following technical solutions.

[0012] A method for preparing a polypeptide-coated quercetin molecular capsule.

[0013] The method comprises:

[0014] (1) Amino acid A, quercetin and water were mixed in proportion and subjected to ultrasonic enzymatic hydrolysis to prepare a precursor;

[0015] (2) Mixing the precursor and amino acid B in proportion to prepare a molecular capsule precursor;

[0016] (3) The molecular capsule precursor is added to the crystallizer to recrystallize the outer layer structure, filtered and dried under high pressure to prepare polypeptide-coated quercetin molecular capsules.

[0017] As a preference,

[0018] The amino acid A in step (1) is L-histidine;

[0019] In step (1), the amino acid A, quercetin and water are mixed in a mass ratio of 0.9-1.2):1:(3-5).

[0020] As a preference,

[0021] The ultrasonic enzymatic hydrolysis process in step (1) uses a neutral protease with an activity of 2000 to 3000 U / g dry basis;

[0022] The amount of the neutral protease used is 8-12 wt% of quercetin.

[0023] As a preference,

[0024] The amino acid B in step (2) is a mixture of β-alanine (D-type) and L-histidine;

[0025] The content of L-histidine in the amino acid B is 28-32 wt %, and the balance is β-alanine (D type).

[0026] As a preference,

[0027] During the preparation of the molecular capsule precursor in step (2), the reaction temperature is controlled at 45-50°C, and the pH value of the reaction system is adjusted to 6.8-7.2. Proteinase K (liquid) with an activity of 600 U / mL is added during the reaction. The amount of proteinase K (liquid) added is 4-6 wt% of amino acid B.

[0028] As a preference,

[0029] The recrystallization process in step (3) is carried out at 30-35°C, the crystallizer speed is controlled to be 300-400 rpm, and the recrystallization process is continued for 1-2 hours.

[0030] As a preference,

[0031] The high-pressure drying is carried out at 24-26° C. and 4-6 MPa for 4-5 hours.

[0032] A polypeptide-encapsulated molecular capsule of quercetin.

[0033] Quercetin reacts directly with superoxide anions and hydroxyl radicals through the single electron transfer of o-diphenolic hydroxyl groups, acting as a hydrogen donor. In this process, quercetin forms more stable intramolecular hydrogen bonds, preventing the peroxidation of unsaturated fatty acids and arachidonic acid, and reducing damage to biological membranes. Quercetin can act on free radical-related enzymes, causing protein precipitation and preventing the synthesis of oxygen free radicals. Quercetin can also chelate with metal ions in cells to inhibit the production of hydroxyl free radicals.

[0034] Nano-drug delivery system is a special dosage form that uses nanoparticles to carry drugs (unless otherwise specified in this invention, the drug specifically refers to quercetin), which can improve various problems existing in drug use. Among them, the nano-drug delivery system based on biofilm has better performance as a biomimetic drug carrier.

[0035] The outer layer of the biomembrane is composed of a phospholipid bilayer, with an inner cavity capable of loading macromolecules, small molecules, and nucleic acids, providing excellent biotransmission and sustained-release properties. However, during use, compatibility issues between the biomembrane and the drug, as well as inherent drug instability, have been found to result in low drug loading rates, uneven loading, and poor stability.

[0036] In contrast, the present invention uses L-carnosine as a base material and utilizes an embedding complexation method to prepare molecular capsules. As a bioactive polypeptide, L-carnosine has broad application prospects in the fields of medicine, cosmetics, health products, and food. Molecular encapsulation technology can also isolate the core material from the surrounding environment, reducing damage to the core material from the external environment (water, light, oxygen, temperature, etc.), thereby improving and enhancing the appearance and properties of the core material, which is beneficial for the storage, transportation, and consumption of these nutrients. The wall membrane also has a sustained-release function, controlling the release rate of the core material's biologically active ingredients to fully exert their effects on the human body. Furthermore, the shielding effect of the wall material can mask the core material's undesirable color, odor, and bitterness. Therefore, this technology has a wide range of applications in the food, medicine, and biology fields.

[0037] In the technical solution of the present invention, the molecular capsule is a special structure with a special three-dimensional cavity formed by two or more molecules connected together by reversible intermolecular non-covalent interactions. It has potential application value in the fields of controlling drug release, catalysis, separation, materials science and biomedicine. The present invention also treats amino acids and quercetin molecules through ultrasonic enzymatic hydrolysis to promote the unfolding speed of amino acid molecules, thereby accelerating the hydrolysis rate. Ultrasonic enzymatic hydrolysis can not only increase the solubility of the precursor and increase the activity of ACE inhibitory peptides, but also generate a large number of precursors with enhanced antioxidant activity. The precursors after ultrasonic treatment can be directly used to prepare hypoallergenic bioactive peptides.

[0038] In the above-mentioned construction process, carnosine itself has the characteristic of binding to quercetin at both ends. Carnosine is a dipeptide whose monomers are L-histidine and β-alanine (D-form). Both L-histidine and β-alanine (D-form) can form intermolecular bonds with quercetin. Therefore, if carnosine is directly used to bind to quercetin, the different volume effects generated by the two monomers will lead to a relatively chaotic actual binding pattern of carnosine, ultimately reducing the drug loading capacity to a certain extent. Moreover, the uneven distribution of the exo-terminal components and active groups will lead to weakened permeability. In actual use, the drug loading capacity is reduced and the controllability of drug release capacity is poor. However, the present invention first uses L-histidine to achieve initial binding with quercetin to form a precursor, and then adds β-alanine (D-form) to complete the reaction construction of carnosine. This can effectively ensure the binding form of carnosine and quercetin, thereby ensuring the stability of the properties and effects of the final product.

[0039] In the molecular capsules constructed, on the one hand, the wall material of the molecular capsule plays a decisive role in the effect, affecting the fluidity, permeability, solubility and sustained-release performance of the encapsulated product. The ideal wall material needs to be compatible with the core material, have good stability, good hygroscopicity and solubility, stable chemical properties, wide source and low price; on the other hand, the molecular capsules based on carnosine have a skeleton with a certain cavity and bonding units that can interact reversibly. Due to the structural characteristics of the carnosine molecule, which is wide at the top and narrow at the bottom and has a "cup-shaped" cavity inside, it is easy to derivatize and introduce specific functional groups, making it an ideal skeleton for constructing supramolecular capsules.

[0040] In the technical solution of the present invention, the outer layer structure is recrystallized, and after modification, the obtained product is spherical, but its agglomeration traces are not messy, but a polycrystalline agglomerate arranged in a radial order. The agglomerate initially forms a filamentous fiber, and then begins to branch and grow from the two ends of the fiber to form a "wheat bundle" crystal that is narrow in the middle and thick at both ends. Finally, through continuous small-angle bifurcation, spherical crystals with a spherical morphology are formed. Using spherical crystallization technology, small-particle needle-shaped quercetin is converted into compact spherical crystals. This conversion process improves the sphericity and particle size of the product, while reducing the electrostatic interaction and friction between crystals. Therefore, this method significantly improves the powder properties of the raw material, such as fluidity, compressibility, and bulk density. Through spherical crystallization technology, spherical microparticles of quercetin coated with carnosine as a carrier are prepared, which can achieve the controlled release of quercetin.

[0041] In the technical solution of the present invention, researchers have successfully prepared a spherulite with specific properties. The entropy value of the spherulite is significantly low, which is conducive to maintaining its highly ordered structure, so that it can better maintain its shape and size. In addition, its melting point is extremely high, which means that it can maintain stable performance in a high-temperature environment and exhibit excellent thermal stability. The solubility and dissolution rate of the spherulite are significantly low, which allows it to be released at a slower rate in the body, so that it can better exert its efficacy. This slow release rate not only improves the bioavailability of the drug, but also effectively reduces side effects, making the drug safer and more effective. In summary, the spherulite prepared by the technical solution of the present invention has become an ideal form of biopharmaceutical preparation with its many advantages such as low entropy value, high melting point, low solubility and dissolution rate, and high bioavailability, and has broad application prospects.

[0042] The present invention is beneficial in that:

[0043] The present invention can effectively improve the bioavailability of quercetin in the constructed molecular capsule by optimizing the powder properties of the drug crystals, and achieves controlled sustained release through specific spherical diameters to better control the release rate and amount of quercetin. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 shows the penetration of moisturizing lotion into the skin;

[0045] Figure 2 shows the penetration of the repairing eye cream into the skin;

[0046] Figure 3 shows the penetration of the essence into the skin;

[0047] Figure 4 shows the drug uptake by cells in the moisturizing lotion control group after 4 h;

[0048] Figure 5 shows the drug uptake by cells in the moisturizing lotion experimental group 4 h later;

[0049] Figure 6 shows the drug uptake by cells in the repair eye cream control group 4 hours later;

[0050] Figure 7 shows the drug uptake by cells in the repair eye cream experimental group 4 hours later;

[0051] Figure 8 shows the drug uptake by cells in the essence control group 4 h later;

[0052] Figure 9 shows the drug uptake by cells in the essence experimental group 4 hours later. Modes for Carrying Out the Invention

[0053] The present invention is further described in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0054] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all methods known to those skilled in the art.

[0055] Example 1

[0056] Preparation method of polypeptide-coated quercetin molecular capsule

[0057] (1) L-histidine, quercetin and distilled water were mixed in a mass ratio of 0.9:1:3, and 2000 U / g (dry basis) neutral protease (8 wt% of quercetin) was used for enzymatic hydrolysis at 43 °C, pH 6.8 and ultrasonic frequency 170 W for 3 h to prepare a precursor;

[0058] (2) The precursor and amino acid B were mixed evenly in a mass ratio of 1:0.8, and reacted at 45 °C and pH 6.8 for 3 h to prepare a molecular capsule precursor. During the process, 600 U / mL of proteinase K was added, which was 4 wt% of the mass of amino acid B. The amino acid B was a mixture of β-alanine (D type) and L-histidine, of which the L-histidine component accounted for 28 wt% and the balance was β-alanine (D type);

[0059] (3) The molecular capsule precursor was added to the crystallizer, and the outer layer structure was recrystallized at a temperature of 30 °C and a rotation speed of 300 rpm for 1 h. The solid phase was retained by filtration and dried at a temperature of 24 °C and a pressure of 6 MPa for 4 h to prepare polypeptide-coated quercetin molecular capsules.

[0060] The prepared polypeptide-coated quercetin molecular capsules were subjected to the following performance tests, and the specific characterization results are as follows.

[0061] Antioxidant activity assay:

[0062] Superoxide anion (O 2- Free Radical Scavenging Rate: Prepare a 0.05 mol / L Tris-HCl solution (pH 8.2) with 0.1 mol / L HCl. Add 4.5 mL of the Tris-HCl solution to 4.2 mL of the molecular capsule solution, mix thoroughly, and react in a 25°C water bath for 20 minutes. Preheat 3 mmol / L pyrogallol in a 25°C water bath. Add 0.3 mL of pyrogallol to the heated reaction solution and immediately measure its absorbance at 300 nm. Measure the absorbance every 30 seconds. The slope of the absorbance over a 4-minute period is used to calculate the superoxide anion radical scavenging rate. The details are shown below.

[0063]

[0064] Where:

[0065] K0——Distilled water comparison table;

[0066] K——Sample.

[0067] Reducing power assay: Prepare 0.2 mol / L phosphate buffer at pH 6.6. Add 2.5 mL of phosphate buffer and 2.5 mL of potassium ferricyanide (1%) to 1 mL of the molecular capsule test solution. Mix thoroughly, incubate in a 50°C water bath for 20 minutes, and terminate the reaction by adding 2.5 mL of trichloroacetic acid (10%). Centrifuge at 5000 rpm for 10 minutes, then remove 2.5 mL of the supernatant, mix with 2.5 mL of distilled water and 0.5 mL of FeCl₃, and let stand for 10 minutes. Measure absorbance at 300 nm; the absorbance value represents the reducing power of the sample.

[0068] Hydroxyl radical (OH·) scavenging rate assay: Prepare test samples a, b, and c according to the following characterization method. Mix 1 mL of each test sample, 6 mmol / L ferrous sulfate, and 6 mmol / L salicylic acid-ethanol solution. Add 1 mL of saturated H2O2 to initiate the reaction. Incubate the reaction in a 37°C water bath for 30 minutes. Measure the absorbance of the reaction solution at 510 nm and calculate the hydroxyl radical (OH·) scavenging rate, as shown below.

[0069]

[0070] Molecular capsule aqueous solution (mL) Ferrous sulfate (mL) Salicylic acid-ethanol solution (mL) Ethanol (mL) Distilled water (mL) Saturated H2O2 (mL) a111--1b11-1-1c-11-11

[0071] Determination of ACE inhibition rate: Prepare 0.1 mol / L phosphate buffer with 0.3 mol / L sodium chloride and adjust the buffer to pH 8.3. Then, prepare a 5 mmol / L HHL solution using the buffer. The amounts of each reagent to be tested are characterized as follows. After the reaction, add 0.4 mL of ethyl acetate to extract the hippuric acid from the reaction solution. Vortex the mixture until homogenized and centrifuge at 4000 rpm for 10 minutes. Transfer 0.3 mL of the supernatant to a clean centrifuge tube. Heat and dry the tube. Cool to room temperature, then add 0.6 mL of ultrapure water. After extracting any sample adhering to the tube wall, measure the absorbance at 228 nm and calculate the ACE inhibition rate. The details are as follows.

[0072]

[0073] Where:

[0074] A I - sample set;

[0075] A II - control group;

[0076] A III——Terminate the reaction group early.

[0077] Molecular capsule aqueous solution (mL) Saturated hydrochloric acid (mL) HHL (mL) ACE (mL) Distilled water (mL) I501500 II001505 III5501550

[0078] The above test results are shown in the following table.

[0079] Superoxide anion (O2-) free radical scavenging rate, % reducing power hydroxyl radical (OH·) scavenging rate, % ACE inhibition rate, % 56.60.3967.865.2

[0080] In addition to the above efficacy tests, the polypeptide-coated quercetin molecular capsules prepared in the examples were also subjected to in vitro release tests, transdermal release tests and in vitro skin penetration experiments.

[0081] The in vitro release test was performed using an RYJ-12 transdermal diffusion instrument. A control group and a test group were set up respectively, and three parallel tests were set up for each group. The control group was an uncoated mixture of quercetin and carnosine (the mass ratio of quercetin and carnosine was 1:1.2), and the test group was the carnosine-coated quercetin molecular capsule prepared in this example. The diffusion cell and the receiving cell used a PBS-anhydrous ethanol mixed solution with a volume ratio of 4:1. The diffusion cell was diluted to 2 mg / mL by using propylene glycol to dilute the control group and the test group. This test used an artificial semipermeable membrane to simulate the in vitro release platform. The artificial semipermeable membrane was placed in the transdermal instrument. 1 mL of the receiving cell solution was aspirated every 1 h, and then the corresponding volume of isotonic solution was added. After the completion of each time point, the solution was placed in an ultraviolet spectrophotometer to detect the corresponding absorbance, and the in vitro release rate was calculated by comparing with the quercetin-carnosine standard curve.

[0082] Time, h04812162024283236control group, %0233245597180838486test group, %0112333425056606365

[0083] The experimental conditions for transdermal release detection were 37°C, the medium used was a mixture of 80% pH = 7.4 isotonic phosphate buffer and 20% anhydrous ethanol, and the experimental instrument used was a RYJ-12 transdermal diffusion instrument. The experiment set up a test group and a control group respectively. The control group was an uncoated mixture of quercetin and carnosine (the mass ratio of quercetin and carnosine was 1:1.2), and the test group was the carnosine-coated quercetin molecular capsule prepared in this example. Three parallels were set up for each group. The abdominal skin of 6-week-old Wistar rats after depilation was used. After respiratory anesthesia, the rats were depilated, killed, dissected, and the skin was removed from the fat layer. The rats were repeatedly rinsed with clean water, cut into appropriate sizes, placed in physiological saline, and stored at 4°C for later use. The drug used in the diffusion cell was diluted with propylene glycol at a concentration of 2 mg / mL, and the addition amount was 1 mL. The receiving cell was a mixture of 80% pH = 7.4 isotonic phosphate buffer and 20% anhydrous ethanol. The skin was placed between the two and clamped in the transdermal instrument. Every corresponding time, 1% of the receiving cell solution was aspirated. mL, and then add the corresponding volume of medium solution; after taking the sample at each time point, place the solution in a UV spectrophotometer to detect the absorbance, compare it with the quercetin standard curve, calculate the concentration, calculate the transmittance, and draw a chart.

[0084] Time, h04812162024283236Control group, %0213843495355565658Test group, %0295369778386899192

[0085] In vitro skin permeation experiments were conducted using fresh abdominal skin from three-month-old miniature pigs. After hair removal, the fat layer and part of the dermis were scraped off with a scraper, and the skin was cut into approximately 3.14 cm 2 The test and control groups were each diluted to 50 μg / mL (as FITC-HA) in pH 7.4 PBS buffer. The control group consisted of an uncoated mixture of quercetin and carnosine (with a quercetin:carnosine mass ratio of 1:1.2). The test group consisted of carnosine-coated quercetin molecular capsules prepared in this example. 0.5 mL of each dilution was added to the permeation device and incubated in the dark for 30 minutes. After removal, the skin was rinsed repeatedly with clean water until the surface fluorescence showed no discoloration. The surface moisture was dried with absorbent paper and incubated in the dark for 10 minutes to dry slightly. The skin was then sectioned and observed under a fluorescence microscope for fluorescence penetration.

[0086] The penetration depth of the control group was 239±56 μm, and the penetration depth of the test group (μm) was 552±21 μm.

[0087] It can be clearly seen from the above in vitro characterization results that the molecular capsules of the present invention have obvious sustained-release characteristics in vitro, and can maintain a relatively stable and slow release within 36 hours, while the control group samples will be released rapidly and concentratedly within about the first 24 hours, indicating that their release controllability is poor. The present invention can use the molecular capsules of the polypeptide-coated quercetin of the present invention for long-term release of ingredients in vitro through the characteristics of stable sustained release to stably provide quercetin. On the other hand, the molecular capsules of the present invention have extremely high transdermal release rates and extremely high release depths. This is mainly based on the polypeptide shell layer, which makes the overall molecular capsule have very good skin penetration ability. Therefore, when the molecular capsules of the present invention are used in skin care products and common cosmetic additives, they can play a role of stable release and rapid penetration and absorption after release, thereby effectively exerting the anti-inflammatory and other abilities of quercetin itself.

[0088] Example 2

[0089] Preparation method of polypeptide-coated quercetin molecular capsule

[0090] (1) L-histidine, quercetin and distilled water were mixed in a mass ratio of 1:1:4, and a neutral protease of 2500 U / g (dry basis) with a mass of 10 wt% of quercetin was used to perform enzymatic hydrolysis at a temperature of 45 °C, a pH of 7 and an ultrasonic frequency of 150 W for 2.5 h to prepare a precursor;

[0091] (2) The precursor and amino acid B were mixed evenly in a mass ratio of 1:1, and reacted at 47 °C and pH 7 for 2.5 h to prepare a molecular capsule precursor. During the process, 600 U / mL of proteinase K was added, which was 5 wt% of the mass of amino acid B. The amino acid B was a mixture of β-alanine (D type) and L-histidine, of which the L-histidine component accounted for 30 wt% and the balance was β-alanine (D type);

[0092] (3) The molecular capsule precursor was added to the crystallizer, and the outer layer structure was recrystallized for 1.5 h at a temperature of 33 °C and a rotation speed of 350 rpm. The solid phase was retained by filtration and dried at a temperature of 25 °C and a pressure of 5 MPa for 4.5 h to prepare polypeptide-coated quercetin molecular capsules.

[0093] The molecular capsules prepared in this example were subjected to the same in vitro release test, transdermal release test and in vitro skin permeation test as in Example 1.

[0094] The test results are shown in the following table.

[0095] Example 3

[0096] Preparation method of polypeptide-coated quercetin molecular capsule

[0097] (1) L-histidine, quercetin and distilled water were mixed in a mass ratio of 1.2:1:5, and 3000 U / g (dry basis) neutral protease with a mass of 12 wt% of quercetin was used for enzymatic hydrolysis at a temperature of 47 °C, a pH of 7.2 and an ultrasonic frequency of 130 W for 2 h to prepare a precursor;

[0098] (2) The precursor and amino acid B were mixed evenly in a mass ratio of 1:1.2, and reacted at 50 °C and pH 7.2 for 2 h to prepare a molecular capsule precursor. During the process, 600 U / mL of proteinase K was added, which was 6 wt% of the mass of amino acid B. The amino acid B was a mixture of β-alanine (D type) and L-histidine, of which the L-histidine component accounted for 32 wt% and the balance was β-alanine (D type);

[0099] (3) The molecular capsule precursor was added to the crystallizer, and the outer layer structure was recrystallized at a temperature of 35 °C and a rotation speed of 400 rpm for 1 h. The solid phase was retained by filtration and dried at a temperature of 26 °C and a pressure of 4 MPa for 5 h to prepare polypeptide-coated quercetin molecular capsules.

[0100] The molecular capsules prepared in this example were subjected to the same in vitro release test, transdermal release test and in vitro skin permeation test as in Example 1.

[0101] The test results are shown in the following table.

[0102] Comparative Example 1

[0103] A method for preparing a polypeptide-coated quercetin molecular capsule. The specific preparation method is the same as that of Example 2, except that the polypeptide coating method of the present invention is changed. The preparation of the polypeptide-coated quercetin molecular capsule is carried out. The specific operations are as follows:

[0104] Preparation method of polypeptide-coated quercetin molecular capsule

[0105] (1) β-alanine (D-type) and L-histidine were mixed in a mass ratio of 1:1, and the amino acids were dissolved in distilled water twice the mass of the amino acid mixture. 2500 U / g (dry basis) neutral protease was added at 5 wt% of the amino acid mass. Ultrasonic enzymatic hydrolysis was performed at a temperature of 45 °C, a pH of 7, and an ultrasonic frequency of 150 W for 2.5 h to prepare a coated precursor.

[0106] (2) The coating precursor and quercetin were mixed evenly at a mass ratio of 4.5:1, and reacted at 45 °C, pH 7, and nitrogen atmosphere for 2 h to prepare a molecular capsule precursor;

[0107] (3) The molecular capsule precursor was added to the crystallizer, and the outer layer structure was recrystallized for 1.5 h at a temperature of 33 °C and a rotation speed of 350 rpm. The solid phase was retained by filtration and dried at a temperature of 25 °C and a pressure of 5 MPa for 4.5 h to prepare polypeptide-coated quercetin molecular capsules.

[0108] The molecular capsules prepared in this example were subjected to the same in vitro release test, transdermal release test and in vitro skin permeation test as in Example 1.

[0109] The test results are shown in the following table.

[0110]

[0111] As can be clearly seen from the table above, the present invention directly encapsulates quercetin with carnosine. Compared with the molecular capsules prepared in Example 2, the encapsulated quercetin exhibits a significant decrease in in vitro release rate and fluctuating release, failing to achieve effective and stable in vitro release. Furthermore, the transdermal release rate and in vitro skin penetration depth also significantly decreased, significantly reducing the actual efficacy of the quercetin and lowering the actual absorption rate of quercetin.

[0112] Comparative Example 2

[0113] A method for preparing a polypeptide-coated quercetin molecular capsule is described. The specific preparation method is the same as that in Example 2, except that the polypeptide coating sequence of the present invention is changed. The specific steps are as follows:

[0114] Preparation method of polypeptide-coated quercetin molecular capsule

[0115] (1) β-Alanine (D-type), L-histidine, quercetin and distilled water were mixed in a mass ratio of 1:1.5:0.6:4 and reacted at 45 °C, pH 7, nitrogen atmosphere and ultrasonic frequency of 150 W for 4 h. During the process, 600 U / mL proteinase K with 5 wt% of amino acid mass and 2500 U / g (dry basis) of neutral protease with 5 wt% of amino acid mass were added to prepare the molecular capsule precursor;

[0116] (2) The molecular capsule precursor was added to the crystallizer, and the outer layer structure was recrystallized for 1.5 h at a temperature of 33 °C and a rotation speed of 350 rpm. The solid phase was retained by filtration and dried at a temperature of 25 °C and a pressure of 5 MPa for 4.5 h to prepare polypeptide-coated quercetin molecular capsules.

[0117] The molecular capsules prepared in this example were subjected to the same in vitro release test, transdermal release test and in vitro skin permeation test as in Example 1.

[0118] The test results are shown in the following table.

[0119]

[0120] It can be clearly seen from the above table that the encapsulation of quercetin is achieved while constructing carnosine, and its actual effect is very close to that of Comparative Example 1. Compared with the molecular capsules prepared in Example 2, it can be seen that its in vitro release rate has decreased and fluctuated, and it cannot be effectively and smoothly released in vitro. In addition, the transdermal release rate and in vitro skin penetration depth have also decreased significantly, resulting in a significant decrease in its actual use effect and a decrease in the actual use absorption rate of quercetin.

[0121] Comparative Example 3

[0122] A commercially available cyclodextrin inclusion quercetin nanoparticle was subjected to the same in vitro release test, transdermal release test and in vitro skin permeation test as in Example 1.

[0123] The test results are shown in the following table.

[0124]

[0125] As can be clearly seen from the table above, the in vitro release of quercetin nanocapsules encapsulated with cyclodextrin exhibits ultra-high efficiency over a 4-12 hour period. However, the release rates in both the early and late stages are very slow, indicating a lack of long-term, stable, and sustained release. This suggests that the nanocapsule's encapsulation efficiency is poor. On the other hand, the transdermal release rate remains consistently low, even lower than that of the control group of the present invention, indicating that cyclodextrin does not effectively promote quercetin transdermal absorption. Similarly, in vitro skin permeation test results also indicate that the penetration depth is far lower than that of the molecular capsules constructed using the present invention's technical solution, which is also related to the differences between the encapsulated carnosine and cyclodextrin.

[0126] Application Examples

[0127] The polypeptide-coated quercetin molecular capsules prepared in Example 2 were used to prepare three products: moisturizing lotion, repairing eye cream, and essence. The specific preparation methods are as follows.

[0128] Preparation of moisturizing lotion:

[0129]

[0130] (1) Mix and stir phase A at 80-85 °C;

[0131] (2) Heat phase B at 80-85°C to mix and dissolve, then add phase B to phase A and stir and homogenize for 5-10 minutes;

[0132] (3) Add phase C to phase AB, stir and mix evenly, homogenize for 5 minutes, stir at low speed and cool to 40-50°C, add phase D ingredients, stir and mix evenly to make a moisturizing lotion.

[0133] Preparation of Repairing Eye Cream:

[0134]

[0135] (1) Mix and stir phase A at 80-85 °C;

[0136] (2) Heat phase B at 80-85°C to mix and dissolve, then add phase B to phase A and stir and homogenize for 5-10 minutes;

[0137] (3) Add phase C to phase AB, stir and mix evenly, homogenize for 5 minutes, stir at low speed and cool to 40-50 °C, add phase D ingredients, stir and mix evenly to make a repairing eye cream.

[0138] Preparation of essence:

[0139]

[0140] (1) Mix the raw materials of phase A at 75-85 °C, stir and mix until a uniform solution is formed;

[0141] (2) Cool down to 40-50 °C, add phase B ingredients and mix well;

[0142] (3) Stir at low speed, add the ingredients of Phase C one by one, stir and mix evenly to make the essence.

[0143] In vitro skin penetration testing: Three-month-old fragrant pig abdominal skin was used. After hair removal and removal of the fat layer and part of the dermis, the skin was cut into circular pieces with a diameter of 400 mm and fixed to a permeation device with the stratum corneum facing upward. FITC-HA was used as the fluorescein for the experiment. After cross-linking quercetin with fluorescein, the drug was loaded using the encapsulation technique described in this example. The product was then diluted to 50 μg / mL (as FITC-HA) using pH 7.4 PBS buffer. 0.5 mL of each of the above dilutions was added to the permeation device and incubated in the dark for 30 minutes. After the test, the skin was removed and repeatedly rinsed with clean water until the surface fluorescence showed no discoloration. The surface moisture was dried with absorbent paper and incubated in the dark for 10 minutes to dry slightly. The skin was then sectioned and observed under a fluorescence microscope for permeation of the fluorescence within the skin. Characterization results are shown in Figures 1-3.

[0144] As can be seen from Figures (1 to 3), the encapsulated quercetin has a strong ability to penetrate the skin. The wider the fluorescent band, the deeper the drug penetration. By comparing the penetration ability of the three cosmetics into the skin surface and dermis, and the uptake ability of quercetin by cells in different skin layers, it was observed through a fluorescence microscope that the encapsulated quercetin can penetrate the epidermis and reach the connective tissue of the dermis, allowing the dermis cells to fully contact the quercetin. Therefore, it can be seen that the coating treatment of quercetin by the technical method of the present invention can effectively improve the penetration of quercetin in the skin.

[0145] Based on the above, further drug cellular uptake assays were conducted: mouse fibroblasts (NCTC clone 929) were used as target cells, fluorescein isothiocyanate (isomer I) was used as the cytosolic fluorescent marker, and the nuclear dye DAPI was used for nuclear staining. After cross-linking quercetin with fluorescein, the quercetin was coated using the method described in the previous example to prepare fluorescent molecular capsules. Phosphate-buffered saline was then used to dissolve the fluorescent-encapsulated drug-treated cells. After fixation, permeabilization, and nuclear staining, an anti-fluorescence mounting medium was added. Cellular drug uptake was imaged using a fluorescence microscope 4 hours later and compared with a control group in which the peptide-coated quercetin molecular capsules were replaced with an equal amount of a quercetin and L-carnosine combination. Characterization results are shown in Figures 4 to 9. Observe Figures 4 to 9. The blue fluorescence in the figure represents the cell nucleus, and the green fluorescence represents the drug uptake. The higher the green fluorescence intensity, the better the uptake. Since it is difficult to show the color in the figure, the spot size can be roughly estimated. When the drug is taken up, the spot size increases, and the more drug accumulates in the cell, that is, the better the cell's ability to take up the drug. As can be seen from the figure, compared with the control group, the cell uptake rate of the polypeptide-coated quercetin molecular capsule of the present invention is much higher than the uptake rate of the quercetin stock solution. Therefore, it can be seen that the polypeptide coating significantly improves the cellular entry ability of quercetin.

Claims

1. A method for preparing a polypeptide-coated quercetin molecular capsule, characterized in that: The method comprises: (1) amino acid A, quercetin and water are mixed in proportion and subjected to ultrasonic enzymatic hydrolysis to prepare a precursor; (2) Mixing the precursor and amino acid B in proportion to prepare a molecular capsule precursor; (3) Adding the molecular capsule precursor into a crystallizer to recrystallize the outer layer structure, filtering and high-pressure drying to prepare polypeptide-coated quercetin molecular capsules.

2. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1, characterized in that: The amino acid A in step (1) is L-histidine; In step (1), the amino acid A, quercetin and water are mixed in a mass ratio of 0.9-1.2):1:(3-5).

3. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1, characterized in that: The ultrasonic enzymatic hydrolysis process in step (1) uses a neutral protease with an activity of 2000 to 3000 U / g dry basis; The amount of the neutral protease used is 8-12 wt % of quercetin.

4. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1, characterized in that: The amino acid B in step (2) is a mixture of β-alanine (D-type) and L-histidine; The content of L-histidine in the amino acid B is 28-32 wt %, and the remainder is β-alanine (D type).

5. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1 or 4, characterized in that: In the process of preparing the molecular capsule precursor in step (2), the reaction temperature is controlled to be 45-50°C, and the pH value of the reaction system is adjusted to 6.8-7.

2. During the reaction, proteinase K (liquid) with an activity of 600 U / mL is added, and the amount of proteinase K (liquid) added is 4-6 wt% of amino acid B.

6. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1, characterized in that: The recrystallization process in step (3) is carried out at 30-35°C, the crystallizer speed is controlled at 300-400 rpm, and the recrystallization continues for 1-2 h.

7. The method for preparing a polypeptide-coated quercetin molecular capsule according to claim 1, characterized in that: The high pressure drying is carried out at 24-26°C and 4-6 MPa for 4-5 hours.

8. A molecular capsule of polypeptide-coated quercetin prepared by the method of any one of claims 1 to 7.

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